U.S. patent number 4,812,405 [Application Number 06/830,304] was granted by the patent office on 1989-03-14 for double auxotrophic mutants of pichia pastoris and methods for preparation.
This patent grant is currently assigned to Phillips Petroleum Company. Invention is credited to Mary E. Digan, Stephen V. Lair.
United States Patent |
4,812,405 |
Lair , et al. |
March 14, 1989 |
Double auxotrophic mutants of Pichia pastoris and methods for
preparation
Abstract
Processes for the preparation of double auxotrophic mutants of
Pichia pastoris are provided, as well as novel double auxotrophic
mutant strains produced thereby.
Inventors: |
Lair; Stephen V. (La Jolla,
CA), Digan; Mary E. (San Diego, CA) |
Assignee: |
Phillips Petroleum Company
(Bartlesville, OK)
|
Family
ID: |
25256712 |
Appl.
No.: |
06/830,304 |
Filed: |
February 18, 1986 |
Current U.S.
Class: |
435/254.23;
435/938 |
Current CPC
Class: |
C12N
1/165 (20210501); C12N 15/04 (20130101); C12N
15/815 (20130101); C12R 2001/84 (20210501); Y10S
435/938 (20130101) |
Current International
Class: |
C12N
15/81 (20060101); C12N 15/04 (20060101); C12N
001/16 (); C12N 015/00 (); C12R 001/84 () |
Field of
Search: |
;435/172.1,172.3,171,224,255,938,190,232 ;935/69,79,97 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Advances in Protoplast Research, pp. 99-104, 113-118, L. Ferenczy
and G. L. Farkas, eds.; Pergamon Press, New York. .
Kuntze et al., Current Genetics (1985) 9, 205-209. .
Cregg et al., Mol. and Cell Biol. (1985) 5, 3376-3385. .
Herrman et al., Amino Acid Biosyntheses and Genetic Reg.,
1983..
|
Primary Examiner: Weimar; Elizabeth C.
Assistant Examiner: Carson; Patricia
Attorney, Agent or Firm: Phillips; J. E.
Claims
That which is claimed is:
1. A yeast cell of the species Pichia pastoris as a host capable of
being transformed with recombinant DNA material, wherein said host
is defective in histidinol dehydrogenase activity and in
argininosuccinate lyase activity.
2. A yeast in accordance with claim 1 wherein said yeast cell is
Pichia pastoris NRRL Y-18017 (PPF1).
Description
This invention relates to the field of recombinant DNA technology.
In one of its aspects, the invention relates to novel yeast
strains. In another aspect, the invention relates to processes for
producing novel yeast strains.
BACKGROUND
Up to now, commercial efforts employing recombinant DNA technology
for producing various polypeptides have centered as Escherichia
coli as a host organism. However, in some situations E. coli may
prove to be unsuitable as a host. For example, E. coli contains a
number of toxic pyrogenic factors that must be eliminated from any
polypeptide useful as a pharmaceutical product. The efficiency with
which this purification can be achieved will, of course, vary with
the particular polypeptide. In addition, the proteolytic activities
of E. coli can seriously limit yields of some useful products.
These and other considerations have led to increased interest in
alternative hosts, in particular, the use of eukaryotic organisms
for the production of polypeptide products is appealing.
The availability of means for the production of polypeptide
products in eukaryotic systems, e.g., yeast, could provide
significant advantages relative to the use of prokaryotic systems
such as E. coli for the production of polypeptides encoded by
recombinant DNA. Yeast has been employed in large scale
fermentations for centuries, as compared to the relatively recent
advent of large scale E. coli fermentations. Yeast can generally be
grown to higher cell densities than bacteria and are readily
adaptable to continuous fermentation processing. In fact, growth of
yeast such as Pichia pastoris to ultra-high cell densities, i.e.,
cell densities in excess of 100 g/L, is disclosed by Wegner in U.S.
Pat. No. 4,414,329 (assigned to Phillips Petroleum Co.). Additional
advantages of yeast hosts include the fact that many critical
functions of the organism, e.g., oxidative phosphorylation, are
located within organelles, and hence are not exposed to the
possible deleterious effects of the organism's production of
polypeptides foreign to the wild-type host cells. As a eukaryotic
organism, yeast may prove capable of glycosylating expressed
polypeptide products where such glycosylation is important to the
bioactivity of the polypeptide product. It is also possible that as
a eukaryotic organism, yeast will exhibit the same codon
preferences as higher organisms, thus tending toward more efficient
production of expression products from mammalian genes or from
complementary DNA (cDNA) obtained by reverse transcription from,
for example, mammalian mRNA.
The development of poorly characterized yeast species as
host/vector systems is severely hampered by the lack of knowledge
about transformation conditions and suitable vectors. In addition,
auxotrophic mutations are often not available, precluding a direct
selection for transformants by auxotrophic complementation.
Moreover, where auxotrophic mutants are available, it is often not
possible to prepare double auxotrophic mutants by mating procedures
since mating protocols are unknown or do not exist for some yeast
strains. Double auxotrophic mutants are desirable host organisms
because they allow a variety of selection pressures and conditions
to be used for strain manipulation.
If recombinant DNA technology is to fully sustain its promise, new
host/vector systems must be devised which facilitate the
manipulation of DNA as well as optimize expression of inserted DNA
sequences so that the desired polypeptide products can be prepared
under controlled conditions and in high yield.
OBJECTS OF THE INVENTION
An object of the present invention, therefore, is the preparation
of novel double auxotrophic mutants of the species Pichia
pastoris.
Another object of the invention is the development of methods for
the preparation of double auxotrophic mutants of yeasts of the
species Pichia pastoris.
These and other objects of the invention will become apparent from
inspection of the disclosure and claims herein provided.
STATEMENT OF THE INVENTION
In accordance with the present invention, there have been developed
processes for the preparation of double auxotrophic mutants of
yeast strains of the species Pichia pastoris.
Further in accordance with the present invention, novel double
auxotrophic mutant strains of microorganisms of the species Pichia
pastoris are provided. Such double auxotrophic mutants have not
previously been available. Double auxotrophic mutants are useful
hosts for transformation with recombinant DNA material.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, yeast cells of the
species Pichia pastoris which are capable of being transformed with
recombinant DNA material are provided. The yeast cells of the
present invention have two distinct defects in their biosynthetic
pathways relative to wild-type cells of the species Pichia
pastoris. The yeast cells of the present invention are useful, in
one respect, as host cells for recombinant DNA material.
Further in accordance with one embodiment of the present invention,
a cell fusion process for producing double auxotrophic mutants of
yeast strains of the species Pichia pastoris is provided. The cell
fusion process of the invention comprises the following steps:
(a) contacting a first and a second monoauxotrophic mutant yeast
strain of the species Pichia pastoris with both a sulfhydryl group
reducing agent and a cell wall degrading reagent under conditions
suitable for the formation and maintenance of spheroplasts;
(b) combining the spheroplasts prepared in accordance with step (a)
to produce a mixture of spheroplasts, then contacting said mixture
of spheroplasts with a cell fusion promoting agent;
(c) incubating the mixture of spheroplasts prepared in accordance
with step (b) under cell wall regenerating conditions to produce a
collection of prototrophic colonies;
(d) plating the prototrophic colonies produced in accordance with
step (c) on presporulation agar and maintaining the resulting
plates at about 30.degree. C. for 12-48 hours;
(e) replica plating the cells produced in accordance with step (d)
onto sporulation agar and maintaining the resulting plates at about
30.degree. C. for 3-5 days;
(f) alternatively
(1) dissecting the 4-spored asci produced in accordance with step
(e); or
(2) removing the cells from the sporulation plates, resuspending
the cells in phosphate buffered media and exhaustively digesting
the suspended cells with a cell wall degrading reagent;
(g) then germinating and growing each spore in rich media at about
25.degree.-35.degree. C. for 2-5 days; and
(h) screening the collection of cells prepared in accordance with
steps (a)-(g) for double auxotrophic mutants; and
(i) isolating the double auxotrophic mutants from the collection of
cells screened in accordance with step (h).
Suitable mono-auxotrophic mutants for use in the process of the
invention are known to those skilled in the art having been
disclosed in U.S. Application Ser. No. 666,579, filed Oct. 30, 1984
by Stroman et al. and assigned to Phillips Petroleum Company, the
disclosure of which is hereby incorporated by reference. Exemplary
mono-auxotrophic mutants include NRRL Y-15851 (GS115), a histidine
requiring mutant, NRRL Y-18041 (GS190), an arginine requiring
mutant, and the like.
To prepare Pichia spheroplastis, the cells are contacted with a
sulfhydryl group reducing agent, such as for example,
dithiothreitol or .beta.-mercaptoethanol and a cell wall degrading
reagent. An example of a specific solution containing a sulfhydryl
group reducing agent is the 0.7% (v/v) .beta.-mercaptoethanol
solution buffer described in the Examples. Enzymatic digestion of
the cell walls is accomplished by contacting the strains to be
spheroplasted with any of the cell wall degrading reagents known to
those of skill in the art, such as for example, Zymolyase (60,000
units/g; Miles Laboratories), Glusulase (Endo Laboratories), and
the like. A wide variety of temperatures, contact times and dosage
levels for cell wall degrading reagents are operable. Generally, in
the range of about 10 .mu.g to 400 .mu.g of Zymolyase or 600-20,000
units of Glusulase per 10 mL of cell suspension are employed for
spheroplast formation. Preferably about 40-200 .mu.g of Zymolyase
or 1,000-5,000 units of Glusulase 10 mL of cell suspension is
employed. Temperature is generally maintained at about 25.degree.
C. or above, but less than about 35.degree. C. Preferably,
temperature is maintained at about 30.degree. C. Contact time is
generally at least about 15 minutes and usually no greater than
about 60 minutes. While many buffered media are suitable, it is
essential that cells to be converted to spheroplasts be suspended
in a buffer which is iso-osmotic with the cells, such as, for
example, 0.7M MgSO.sub.4, or SCE buffer (sorbitol/citrate/EDTA; see
Examples for recipe).
Spheroplasts of a first and a second monoauxotrophic strain of
Pichia pastoris are prepared as described above, centrifuged, and
the spheroplast pellets are then resuspended in the presence of a
cell fusion promoting agent, e.g., about 1 mL of the spheroplast
fusion media (CaCl.sub.2, polyethylene glycol, DMSO solution) per
spheroplast-containing pellet. The two spheroplast suspensions are
mixed, and maintained at about 20.degree.-30.degree. C. for 5-30
minutes.
The mixed spheroplasts are then treated under cell wall
regenerating conditions. Cell wall regenerating conditions comprise
plating spheroplasts on to plates containing regeneration agar as
medium. A typical regeneration agar provides a balanced osmotic
media and comprises:
MgSO.sub.4 --about 0.35M
dextrose--about 0.1M
yeast nitrogen base--about 7 g/L
Bacto-agar--about 3%
Plates are then incubated--about 25.degree.-35.degree. C. for about
3-5 days.
Only prototrophs can grow on the regeneration agar-containing
plates; parental auxotrophic strains are incapable of growth on
this medium and thus serve as an internal control. The frequency of
recovery of prototrophs was very low on the control plates, which
contained either of the mono-auxotrophic parents, when compared to
the experimental plates, which contained both of the
mono-auxotrophic parents. The prototrophs were transferred to
minimal master plates and then sporulated by transferring them to
presporulation plates for 24 hours and then transferring to
sporulation plates and incubating the plates for 3-5 days. The
resulting 4-spored asci are either dissected, or cells are removed
from sporulation plates, resuspended in phoshate buffered media and
exhaustively digested with a cell wall degrading reagent. As a
result of the latter treatment, vegetative cells are destroyed, and
only random spores remain to be germinated and grown. While the
latter method is preferred for rapid sample generation, the former
method (i.e., dissection of the 4-spored asci) is preferred when a
statistical population of segregants is desired. The resulting
colonies are finally screened for the presence of double
auxotrophic mutants.
The screening of the individual spores derived from the fused cells
is carried out as follows. The spores are germinated and grown up
on rich media at about 25.degree.-35.degree. C. for about 2-5 days,
then plated on a master plate, from which replicate plates are
prepared. One replicate plate contains as medium a minimal media
plus the biosynthetic product in which the first mono-auxotrophic
mutant is defective, while another replicate plate contains as
medium a minimal media plus the biosynthetic product in which the
second mono-auxotrophic mutant is defective. The double auxotrophic
mutants are identified as those cells which are capable of growth
only on rich medium but which are not capable of growth on either
of the minimal media supplemented with only one of the biosynthetic
products in which the mono-auxotrophic strains were deficient. The
colonies that correspond on the master plate to those cells which
did not grow under minimal feed conditions but which did grow on
rich medium were then picked for further characterization.
In accordance with another embodiment of the present invention, a
mating process for producing double auxotrophic mutants of yeast
strains of the species Pichia pastoris is provided. The mating
process of the invention comprises the following steps:
(a) suspending together in rich media colonies of a first and a
second mono-auxotrophic mutant yeast strain of the species Pichia
pastoris ;
(b) plating the suspension containing said first and second
mono-auxotrophic mutant yeast strains prepared in accordance with
step (a) on presporulation agar and maintaining plates at about
30.degree. C. for about 12-48 hours, preferably about 24 hours;
(c) replica plating the cells produced in accordance with step (b)
onto sporulation agar and maintaining the resulting plates at about
30.degree. C. for about 8-48 hours, preferably about 24 hours;
(d) replica plating the cells produced in accordance with step (c)
onto minimal agar and maintaining at 25.degree.-35.degree. C. for
1-5 days, or until colonies are visible;
(e) suspending individual prototrophic colonies obtained from step
(d) in rich media;
(f) plating the suspension produced in accordance with step (e) on
presporulation agar and maintaining the resulting plates at about
30.degree. C. for about 12-48 hours;
(g) replica plating the cells produced in accordance with step (f)
onto sporulation agar and maintaining the resulting plates at about
30.degree. C. for 3-5 days;
(h) alternatively (1) dissecting the 4-spored asci produced in
accordance with step (g), or (2) removing the cells from the
sporulation plates, resuspending the cells in phosphate buffered
media and exhaustively digesting the suspended cells with a cell
wall degrading reagent;
(i) then germinating and growing each spore in rich media at about
25.degree.-35.degree. C. for about 48 hours; and thereafter
(j) screening the colonies prepared in accordance with step (i) for
double auxotrophic mutants.
Suitable mono-auxotrophic mutants for use in the process of the
invention are known to those skilled in the art having been
disclosed in U.S. Application Ser. No. 666,579, filed Oct. 30, 1984
by Stroman et al. and assigned to Phillips Petroleum Company, the
disclosure of which is hereby incorporated by reference. Exemplary
mono-auxotrophic mutants include NRRL Y-15851 (GS115), a histidine
requiring mutant, NRRL Y-18014 (GS190), an arginine requiring
mutant, and the like.
The cells to be mated are first suspended in a rich media, such as
for example, YPD (see examples for recipe) and other similar media
as well known to those of skill in the art. The suspended cells are
plated on presporulation agar, and maintained at about 30.degree.
C. for 12 up to 48 hours. A typical formulation for presporulation
agar is set forth below in the example section.
Once grown on presporulation agar, cells are replica plated to
sporulation agar, then maintained at about 30.degree. C. for about
8-24 hours. A typical sporulation medium is set forth below in the
example section.
Cells grown on the sporulation agar are then replica plated and
germinated on minimal agar at about 25.degree.-35.degree. C. for
about 1-5 days. A typical formulation for minimal agar is:
1-8% dextrose
0.1-2% yeast nitrogen base, minus amino acids, and
1.5-4% agar.
A preferred formulation for minimal agar is set forth in the
examples.
The resultant colonies are suspended in rich media, such as, for
example, YPD, plated on presporulation agar as described
hereinabove and maintained at about 30.degree. C. for about 12-48
hours, then replica plated onto sporulation agar and maintained at
about 30.degree. C. for about 3-5 days. The resulting 4-spored asci
are either dissected, or cells are removed from sporulation plates,
resuspended in phosphate buffered media and exhaustively digested
with a cell wall degrading reagent. As a result of the latter
treatment, vegetative cells are destroyed, and only random spores
remain to be germinated and grown. While the latter method is
preferred for rapid sample generation, the former method (i.e.,
dissection of the 4-spored asxi) is preferred when a statistical
population of segregants is desired. The resulting colonies are
finally screened for the presence of double auxotrophic
mutants.
The screening procedure involves germinating and growing the spores
up on rich medium at about 25.degree.-35.degree. C. for about 3-5
days, then replica plating onto two different plates, one plate
containing synthetic complete media less the bio-synthetic product
in which the first mono-auxotrophic mutant is defective, while the
other plate contains synthetic complete media less the biosynthetic
product in which the second mono-auxotrophic mutant is defective.
The double auxotrophic mutants are identified as those colonies
which grow on the rich media, but are incapable of growth on either
of the synthetic complete media lacking the biosynthetic products
in which either of the mono-auxotrophic mutants, from which the
double auxotroph is derived, is defective.
Alternatively, the screening procedure can entail replica plating
the colonies grown on rich media onto two different plates, the
first of which contains minimal media plus the biosynthetic product
in which the first monoauxotrophic mutant is defective and the
second plate contains minimal media plus the biosynthetic product
in which the second mono-auxotrophic mutant is defective. As in the
previously described screening procedure, the double auxotrophic
mutants are identified as those colonies which grow on the rich
media, but do not grow on either of the supplemented minimal media
plates.
The production of double auxotrophic mutants of Pichia pastoris
provides useful hosts for the production of recombinant DNA
products by yeast. The existence of two auxotrophic mutations in
the same cell allows transformation with transforming DNA which
includes a variety of marker genes, as well as allowing the use of
a variety of selective growth conditions to facilitate
identification and isolation of the desired transformants.
The invention will now be described in greater detail by reference
to the following non-limiting examples.
EXAMPLES
The buffers and solutions employed in the following examples have
the composition given below:
______________________________________ 1 --M Tris buffer 121.1 g
Tris base in 800 mL of H.sub.2 O; adjust pH to the desired value by
adding concentrated (35%) aqueous HCl; allow solution to cool to
room temperature before final pH adjustment; dilute to a final
volume of 1 L. Appropriate dilutions are made from this stock
solution. YPD Medium 1% Bacto-yeast extract 2% Bacto-peptone 2%
Dextrose SCE Buffer 9.1 g Sorbitol 1.47 g Sodium citrate 0.168 g
EDTA 50 mL H.sub.2 O pH to 5.8 with HCl Spheroplast Fusion 30% w/v
PEG8000 Medium 15% w/v DMSO 100 m --M CaCl.sub.2 Regeneration Agar
0.35 --M MgSO.sub.4 2% dextrose 0.675% yeast nitrogen base (minus
amino acids) 3% bacto-agar YEPD Agar 2% dextrose 2% peptone 1%
yeast extract 2% agar Presporulation Agar 5% dextrose (GNAP) 2%
peptone 1% yeast extract 0.5% agar 2.3% nutrient agar Sporulation
Agar 0.5% sodium acetate (anhydrous) 1% KCl 2% agar Minimal Agar 2%
dextrose 0.675% yeast nitrogen base (minus amino acids) 2% agar SC
(synthetic complete) 2% dextrose Agar 0.675% yeast nitrogen base
(minus amino acids) 2% agar amino acid suplementation as follows:
______________________________________ Final Supplemented
Concentration Amino Acid mg/L
______________________________________ adenine sulfate 20 uracil 20
L-tryptophan 20 L-histidine-HCl 20 L-arginine-HCl 20 L-methionine
20 L-tyrosine 30 L-leucine 30 L-isoleucine 30 L-lysine-HCl 30
L-phenylalanine 50 L-glutamic acid 100 L-aspartic acid 100 L-valine
150 L-threonine 200 L-serine 375
______________________________________ SC-arg Agar As SC agar, but
no arginine amino acid supplement SC-his Agar As SC agar, but no
histidine amino acid supplement
______________________________________
EXAMPLE I Pichia pastoris Fusion Procedure
A. Cell Growth
1a. A colony of Pichia pastoris GS115 (NRRL Y-1581) was inoculated
at about 10 mL of YPD medium and shake cultured at 30.degree. C.
for 12-20 hrs.
1b. A colony of Pichia pastoris GS190 NRRL Y-18014) was inoculated
into about 10mL of YPD medium and shake cultured at 30.degree. C.
for 12-20 hours.
2. Cultures were harvested when OD.sub.600 was about 0.2-0.3 (after
approximately 16-20 hrs) by centrifugation at 1500 g for 5
minutes.
B. Preparation of Speroplasts
1. Cells were washed once in 10 mL of sterile water by
centrifugation at 1500 g for 5 minutes.
2. The cells were resuspended in 10 mL of 50 mM tris-HCl, pH 7.5,
0.7M MgSO.sub.4.
3. 200 .mu.L glusulase (to give a final concentration of 2% v/v)
and 10 .mu.L .beta.-mercaptoethanol (to give a final concentration
of 0.7% v/v) were added.
4. Cells were incubated at 30.degree. C. for 90 minutes with gentle
shaking.
Since the preparation of spheroplasts is a critical step in the
fusion procedure, spheroplast formation was monitored as follows:
100 .mu.L aliquots of cells were added to 900 .mu.L of 1M Sorbitol
before or just after the addition of glusulase and at various times
during the incubation period. The incubation was stopped at the
point where cells lyse in SDS but not in sorbitol.
5. Intact cells were separated by low speed centrifugation at 400g
for 5 minutes. The supernatant containing the spheroplasts was then
transferred to fresh tubes.
6. The spheroplast suspension was diluted with 50 mM Tris-HCl, pH
7.5, to decrease the MgSO.sub.4 concentration to 0.25M.
C. Fusions of spheroplastis
1. Spheroplasts were centrifuged at 1000g for 5 minutes and
resuspended in 1 mL of spheroplast fusion medium.
2. A 0.5 mL volume of each mono-auxotrophic spheroplast preparation
was mixed together and the volume in the original tubes was brought
back to 1 mL with spheroplast fusion medium. The tubes with the
mono-auxotropic spheroplasts served as controls for reversion to
prototrophy.
3. All tubes were incubated for 30 minutes at room temperature.
D. Plating of Fusion Samples
1. 20 mL regeneration agar per plate was poured at least 2 hours
before the fusion samples were ready.
2. 1:10 and 1:100 dilutions of each sample were prepared in 1 mL
volumes of the PEG solution. 0.1 mL volumes from each tube/plate
were used for regeneration of spheroplasts.
3. Plates were incubated at 30.degree. C. for 3-5 days.
E. Screening of Cell Fusions
The fused cells obtained employing the above fusion procedure were
screened, and double auxotrophic mutants isolated therefrom as
follows:
After 3-5 days growth on regeneration agar, prototrophic colonies
resulting from the fusions were transferred to minimal medium
master plates. The master plates were incubated for 2 days and
isolates that regrew on the master plates were sporulated by
replica plating on to presporulation plates for 24 hours and then
on to sporulation plates which were incubated at 30.degree. C. for
5 days. The 4-spored asci that resulted were isolated and dissected
by micromanipulation on YPD plates. Alternatively, instead of
dissecting the 4-spore asci, the cells can be removed from the
sporulation plates, washed twice with 0.1M sodium phosphate buffer,
pH 7.5 and resuspended in 2.7 mL of sodium phosphate buffer. To the
suspension was added 3 .mu.L .beta.-mercaptoethanol (0.1% final),
60 .mu.L glusulase (2% final), 300 .mu.L of a zymolayse 60,000
stock solution containing 5 mg/mL in phosphate buffer (0.5 mg/mL
final) and the mixture was digested for 5 hours at 30.degree. C.
with occasional shaking. The resultant spore preparation was
sonicated three times for 15 seconds and harvested by
centrifugation at 3000.times.g for 10 minutes. The resultant pellet
was washed twice with a 0.2% Tween 80 solution and resuspended in
0.1M phosphate buffer at approximately 10.sup.7 spores/mL. Single
spores were germinated after plating on YEPD agar plates. In some
cases, due to clumping of the spores, separation into single spores
was accomplished using a micromanipulator.
The YEPD agar plates were incubated until colonies from the spores
reached sufficient size to be transferred to duplicate YEPD master
plates. One of each YEPD master was then used for replica plating
to synthetic complete medium plates lacking one of the single
auxotrophic requirements (e.g. synthetic complete medium minus
arginine and synthetic complete medium minus histidine). The
results of this screen were used to identify prototrophs by their
growth on both plates; mono-auxotrophs grew on only one of the
plates, and double-auxotrophs failed to grow on either of the
plates. Double auxotrophic isolates were then transferred to YEPD
or synthetic complete plates for storage.
The fusion procedure described hereinabove utilizing the
mono-auxotrophic mutants P. pastoris GS115 (his4) and P. pastoris
GS190 (arg4), led to the isolation of a double auxotrophic mutant
having the phenotype arg.sup.- his.sup.-. This double auxotrophic
mutant has been given the laboratory designation PPF1. P. pastoris
strain PPF1 has been deposited with the Northern Regional Research
Center of the United States Department of Agriculture in Peoria,
Illinois to ensure public access to the strain upon issuance of
this application as a patent. Strain PPF1 has been given the
accession number NRRL Y-18017.
EXAMPLE II
Cross Mating of Pichia pastoris Auxotrophs
The mono-auxotrophic strains GS115 (his4.sup.-) and GS190
(arg4.sup.-) were crossed as follows:
Single colonies of each of the two auxotrophic mutants were
resuspended in 100 .mu.L of YPD, mixed thoroughly, and spread on
presporulation (GNAP) plates. Twenty-four hours after plating on
GNAP, cells were replica-plated onto sporulation agar. Eight hours
after plating on sporulation agar, cells were replica-plated onto
minimal media. These plates were incubated at 30.degree. C. for
1.5-2 days, until colonies were seen.
Prototrophic colonies from minimal plates were sporulated by
subjecting them to a regimen similar to that described above for
mating, e.g., a single colony was dispersed in YPD, plated on GNAP,
and replica-plated onto sporulation agar after 24 hours on GNAP.
After 3 days on sporulation agar, 4-spored asci were dissected on
YEPD plates which then were put at 30.degree. C. to germinate.
Alternatively, the exhaustive digestion procedure for spore
preparation described above can be employed instead of the
dissection procedure. Wild type segregants, as well as single and
double auxotrophs, were identified by replica-plating onto SC-arg
and SC-his media.
The examples have been provided merely to illustrate the practice
of the invention and should not be read so as to limit the scope of
the invention or the appended claims in any way. Reasonable
variations and modifications, not departing from the essence and
spirit of the invention, are contemplated to be within the scope of
patent protection desired and sought.
* * * * *